The feline major histocompatibility complex (MHC) has been studied using molecular techniques as an approach to comparative genome organization of this important gene cluster. The MHC in most mammals consists of two classes of genes, classes I and II, which play special roles in presenting antigens to T-cell receptors. Sequence analysis of eight different domestic cat MHC (FLA) class I cDNA clones revealed strong conservation of invariant and variant amino acid residues in antigen-binding and T-cell recognition sites among human, mouse, and domestic cat. This evidence indicates that the domestic cat MHC class I molecule has a similar structure to human and mouse class I molecules, and its MHC class I molecules have highly polymorphic features. Further analysis revealed the participation of four major factors in the evolution of the feline MHC class I genes. These include: (1) a gradual accumulation of spontaneous mutational substitution, (2) negative selection for functional constraints of class I genes, (3) positive selection in favor of persistence of polymorphism, and (4) periodic intragenic and intergenic DNA recombination. Sequence analysis of MHC class I molecules from two other feline species (cheetah and ocelot) showed that feline MHC class I molecules have highly mosaic structures in their entire coding regions. We identify at least 15 mosaic regions where each has at least two polymorphic sequence motifs. Some of these sequence motifs are conserved not only in Felidae MHC class I sequences but also in some of human, orangutan, and bovine MHC class I sequences. These data revealed that modern polymorphic sequence motifs found in mammalian MHC class I molecules are extremely ancient and shuffling of these motifs by DNA recombination plays a pivotal role in producing novel polymorphic MHC class I molecules in mammals.